Project Summary. Gram-negative (Gram (-)) bacteria are intrinsically resistant to drugs, due to a double membrane structure that acts as a permeability barrier to drugs and as an anchor for efflux pumps. Many Gram (-) bacteria have developed multi-drug resistance, which poses one of the most pressing issues in modern medicine. Antibiotics are barred and extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect. While efforts have focused on targeting one membrane protein at a time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit biosynthesis of multiple membrane proteins simultaneously, thus reducing drug barrier and efflux and accelerating bactericidal action. TrmD is a bacteria-specific S-adenosyl-methionine (AdoMet)- dependent methyl transferase that controls accuracy of the protein-synthesis reading frame. Loss of TrmD increases +1 frameshifts and terminates protein synthesis prematurely. We have discovered that genes for multiple membrane proteins and efflux pumps in E. coli and other Gram (-) bacteria contain TrmD-dependent codons near the start of the reading frame. We hypothesize that targeting TrmD will reduce protein synthesis of all of these genes. By reducing multiple membrane- and efflux-proteins at once, we propose that targeting TrmD offers a novel solution to an unmet need. While AstraZeneca (AZ) has attempted to target TrmD, the isolated hits lacked the cell-permeability needed to exhibit an antibacterial effect. We hypothesize that successful targeting must identify compounds that are cell-permeable and selective for TrmD over the human counterpart Trm5. To test this hypothesis, we have developed and optimized a cell-based fluorescence assay for E. coli TrmD (EcTrmD), in which we will mix a 1:1 ratio of an E. coli mCherry (mCh)-expressing strain dependent on TrmD for survival and a separate YFP-expressing strain dependent on Trm5 for survival to discover cell-permeable compounds that selectively inhibit the TrmD-dependent but not the Trm5-dependent strain. In Aim 1, we will use this cell-based assay, which is high-throughput screening (HTS)-ready, in a large- scale campaign to discover cell-permeable and selective inhibitors of EcTrmD. We will screen a diverse collection of ~180,000 compounds and a collection of 10,000 natural products to identify inhibitors and remove false positives. In Aim 2, we will assess hits in secondary assays to determine their potency and mechanism of action. We will fractionate natural products to active compounds. We will also test hits on Gram (-) bacteria Salmonella and Pseudomonas aeruginosa. In Aim 3, we will use whole-cell assays to identify hits that inhibit cell growth and display TrmD-deficient phenotypes. We will assess initial structure-activity relationship (SAR) of each cluster of hits by analysis of ~20 analogs selected from in silico modeling in our TrmD crystal structure with a bound tRNA and sinefungin (non-reactive AdoMet analog). These initial hits will serve as powerful probes in a new paradigm of antibiotic discovery that inhibits the drug barrier and efflux of Gram (-) bacteria.